Re: fluorescence in situ zymography

From:

"C. H."

Hi, Abigail
Heating that much is probably a bad idea. If you check your spec sheet it
may tell you that PMSF's melting point is about 92-93C. (I just checked the
Sigma-Aldrich website). From what I remember about PMSF, it is typically
dissolved in methanol or DMSO. Don't know for sure if it would interfere
with your experiment. My feeling is that if you add a very small amount of
concentrated PMSF stock solution to your buffer it wouldn't have much of an
affect on your protocol. I know it is used this way for receptor binding
assays without interference.
Cyrla
----Original Message Follows----
From: "Abigail.Mackey"
To: "'HistoNet@pathology.swmed.edu'"
Subject: fluorescence in situ zymography
Date: Thu, 06 Dec 2001 17:58:12 +0000
Hello All,
One other issue with this method to resolve, hopefully the last one!
I need some advice on the use of PMSF(phenylmethylsulphonylfluoride) in
solution. The solution contains:
50mmol Tris-HCl, pH 7.4,
10mmol CaCl2*2H2O,
0.05% Brij 35,
5mmol PMSF
Fluorescence-conjugated gelatin is then added to this solution and incubated
with tissue sections to detect gelatinase activity. As I understand it, PMSF
is added to inhibit any serine proteases which may be present in the tissue.
To get the PMSF to dissolve, the solution needs to be heated to almost
boiling. My first question is will the heating destroy the PMSF action? If
so how do I get it to dissolve another way, can it be dissolved in ethanol
for example? Will the addition of alcohol have any other effects on my
method?
thanks in advance,
abigail
_________________________________
Abigail Mackey
Department Sport and Exercise Sciences
University of Limerick
Ireland
tel +353 (0)61 20 28 84
fax +353 (0)61 33 04 31
_________________________________________________________________
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